この作品は、米軍グラント（W81XWH-08-1-0701）とカール財団病院からフェローシップによって資金を供給されました。

手順はIACUCプロトコル番号13302との完全な互換性であり、動物資源の部門によって承認されています。 1.機器の準備<ol><li>無菌性を確保するために、外科手術の前に使用されるすべての非使い捨て器具をオートクレーブ。 </li><li>使用の間、ガラスビーズ滅菌器とのセッションで複数回使用される器具を滅菌します。 </li></ol> 2.ハイドロゲルの調製<ol><li>解凍し、ヒドロゲル構築物の作成に少なくとも24時間前にプレート幹細胞。 </li><li>以前の刊行物8,18に完全に詳細に記載されているステレオリソグラフィー装置を用いて、外科的処置（または類似の器具）を一日前にパッチを準備します。 </li><li>ハイドロゲルを作成するには、第1の所望のパッチのコンピュータ支援設計（CAD）モデルを生成し、立体造形形式（STL）に設計をエクスポートします。 </li><li> dissolvinによってプレゲル溶液を作成します滅菌1×PBS中のGポリ（エチレングリコール）ジメタクリレート（PEGDMA）は20％のw / v溶液を得ました。同時に、光開始剤、1- [4-（2-ヒドロキシエトキシ） - フェニル] -2-ヒドロキシ-2-メチル-1-プロパン-1-オンDMSO中に溶解させます。 0.5％w / vの最終でPEGDMA溶液に光開始剤を追加します。直接架橋の前に、細胞の所望の密度の100ミリリットルを追加します。注：2.0×10 5 -2.0×10 6細胞/ mlの間の細胞密度が推奨されます。 </li><li> SLAプラットフォームの中央に皿にプレ - ゲル溶液をピペットで、以前にアップロードしたSTLフォーマットされたデザインで、SLAのソフトウェアを実行します。 </li><li>細胞を調節するために可能にするために装置からパッチを取り外し、ダルベッコ改変イーグル培地中でヒドロゲルO / Nインキュベート（DMEM）、5％ウシ胎児血清（FBS）を補充した、100 U / mlペニシリン、および100mg / mlストレプトマイシンパッチ内の環境に。 </li></ol> 3.動物の準備とオーラルintubatiオン<ol><li>ケージの仲間とのグルーミング習慣との相互作用から決定されるような動物は、健康であることを確認する手順の前に、各マウスを評価します。注：女性8-10週齢のC57BL / 6Jマウスは、この手順のために使用された、しかし、任意のマウスの年齢やタイプを使用することができます。 </li><li>麻酔室に動物を置き、1リットル/分O 2をサポートして5％のイソフルランに公開します。つま先ピンチ反射によって麻酔のレベルを監視します。 </li><li>動物を適切に麻酔された後、計量し、挿管台の上に置きます。 </li><li>動物の胸腔に向けて光源を直接および喉頭面を撤回し、声帯を公開するために鉗子を使用しています。慎重に声帯の間の20のG血管カテーテルをガイドし、スムーズに気管に挿入します。カテーテルは小動物人工呼吸器に接続されると、胸腔の機械的な動きによって適切な挿入を検出します。 </li><li>目に換気設定を調整メーカーのガイドラインに基づいて、動物の電子体重。 </li><li>挿管後、低体温症を防ぐために加熱パッド上に仰臥位で動物を配置します。加熱パッドは40℃を超えないことを確認してください。必要に応じて、火傷の可能性を最小限にするために、動物や加熱パッドとの間の障壁を配置します。 </li><li>脱毛クリームを使用することにより、手術部位から髪を削除します。適切な滅菌を確実にするためにベタジンと75％エタノールの3交互スクラブしてサイトを滅菌します。ただ、手術部位を露出させるためにドレープを置き、手順の間に乾燥を防ぐために、目に獣医軟膏を配置します。 </li></ol> 4.手術手順<ol><li>開胸したがって、胸骨の長さを実行する胸骨の左側に皮膚切開約1cmを行い、動物の左側に実行されます。 </li><li>二段階で、基礎となる胸郭から筋層を分離するために鉗子を使用してください。 delineを観察動物の大胸筋を表すレーティングライン。 、少しこの筋肉を持ち上げ、基礎となる外腹斜筋から分離し、内側に後退。そして、同様に、基礎となる胸郭から外腹斜筋を解放し、第2、第3、第4肋骨の暗くされていないビューを提供する、横方向に後退させます。 </li><li>第三及び第四肋骨の間開胸術を行います。静か第4肋骨を持ち上げ第3および第4肋骨の間胸腔を開くために焼灼器を使用しています。さらにキャビティを開き、心臓を露出させるためにリトラクターを配置します。 </li><li>心の薄い心膜を破裂させるために鉗子を使用してください。 </li><li> 8-0モノフィラメントナイロン縫合糸で左冠動脈を結紮。直接左心房の下部先端の下、約4mm心尖から縫合糸を配置します。心室筋のブランチングとスツ語の後に左心房の大きさの増加により、適切な縫合糸の配置を決定します再結ばれています。 </li></ol> 5.パッチの配置とグルー<ol><li>使用するまで37℃でパッチを維持し、5％CO 2の条件。 </li><li>ゆっくりフラットエンドへらを使用してパッチを持ち上げ、ゆっくりと梗塞部位で、心臓の表面の上に置きます。 注：左心室の脱落防止のために、パッチが軽くへら又はピンセットの先端のいずれかを使用して接触を維持することによって所定の位置に保持することができます。 </li><li>トロンビンへのフィブリノゲンの1の比率：4を混合して接着剤を準備します。それは準備の開始後、通常1分以内に、厚く始めるまで繰り返しピペッティングにより溶液を混合。 </li><li>溶液粘度が所望のレベルに達すると、すぐにパッチ表面に約10 mlで移します。注：フィブリン糊の凝固時間は迅速であり、接着剤の効率的な転送のための機会の小窓を提供します。 </li></ol> 6.縫合<ol><l私は&gt; 6-0モノフィラメントナイロン縫合糸を使用して、すべての閉鎖縫合糸を実行します。 </li><li>三から四個々の結節縫合でリブ層を閉じます。 </li><li>肋間層の完全閉鎖する前に、閉鎖が完了した後、胸腔を排気し、適切な胸腔内圧を再確立するために切開部にPE-10カニューレを挿入します。 </li><li>三から四個々の結節縫合で胸筋を閉じます。 </li><li>連続縫合で皮膚層を封止します。 </li><li>完全閉鎖後、カニューレの端に1.0ミリリットルの注射器を接続して、胸腔を排出するためにそれを使用。 </li><li>縫合部位を補強するために切開部位に組織接着剤を適用します。 </li></ol> 7.手術後の処置<ol><li>マウスにブプレノルフィン（0.05〜1.0ミリグラム/ kg）および皮下直前復活に冠動脈結紮および少なくとも20分以下のカルプロフェン（2.2ミリグラム/ kg）を注射を与えます。両薬剤を手術後6-8時間の管理、その後痛みや苦痛を制御するために最大3日間、1日2回与えます。 </li><li>継続的に手術後の最初の4時間を意識し、すべての時間になるまで動物を監視します。鎮痛剤の第二の用量を投与するために、第2の検診を手術（2-4時間後）の夜を実行します。 </li><li>動物が安定した表示されるまで、手順に従って、二日後に毎日動物を監視します。 </li></ol>心機能と組織学の8解析<ol><li>マウスのechocardiographs MI後の損傷の程度を決定するための手順を次の4週間を実行します。 </li><li>吸入麻酔の誘導のための麻酔チャンバー内の4〜5％イソフルラン、およびメンテナンス麻酔用フェイスマスクを介した後、2％イソフルランでマウスを麻酔。 </li><li>脱毛クリームで撮像領域をクリアし、拍動する心臓の2D Mモード心エコー検査スキャンを得るために、小動物の超音波を使用して、機能パラメータのASSOを記録それにciated。 </li><li>その後、24時間、10％ホルマリン溶液中で動物と場所からの心臓を集め、二酸化炭素チャンバーにマウスを安楽死させます。 24時間後、70％エタノールに心を転送し、組織処理まで4℃で維持。 </li><li>心を処理し、パラフィンブロックでそれらを埋め込みます。 </li><li>頂点で始まり、心房で終わる、心臓の心室領域を介して、5mmのセクションをカット。 </li><li>染色は、標準的なプロトコルを使用して、マッソン三色、ヘマトキシリン​​およびエオシン染色の両方でスライスを切断しました。 </li></ol>

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The authors declare that they have no competing financial interests.

マウスMIモデルにこの方法では、我々は他のマウスのMI技術と関連している非心筋領域へのダメージを最小限に抑えるシステムを開発しました。これらの領域は、気管切開による損傷、筋層の切断、および胸腔を露出させるために、リブの破損を含みます。我々は、これらの変更は、外科手術中など、肋骨や筋肉の層を含む、主要な構造の多くの無傷?...

心筋梗塞（MI）は、主要な冠動脈の閉塞によって引き起こされる心臓の領域への血液の中断として定義されます。 MIに起因する損傷が原因または、より具体的には、左心室は、適切に打つ心臓の能力を低下させる非機能的瘢痕組織に生存可能心臓組織のリモデリングです。これは、一回拍出量としても知られているすべてのハートビート、および駆出率6と呼ばれる各心拍で心臓から送り出される血液の割合で身体に送達することができる血液の量の減少をもたらします。これらは、他の減少の機能とともに、十分な機能を維持するために、心臓の残りの部分に負担を増加させます。多くの場合、これは、株は、それが第二の心臓発作、個人7の約10％に見られる現象を引き起こすほど重症になることができます増加しました。 医療行為は、治療のために進化してきたがMIの直後には、何の技術は遅く、停止、ま​​たは組織リモデリングの負の副作用を逆転するように開発されていません。幹細胞療法は、それらの有望な潜在的にもかかわらず、しかし、そのような治療のための可能な道として出現した幹細胞は、臨床設定において成功することが証明されていません。その欠点の一つの理論は、有益な細胞が良好な結果5を生成するのに十分な長さ梗塞部位に残って確保することができないことです。これは単に、梗塞の部位に注射された細胞のない24％以上が生存していないと損傷部位1日後に配達2にとどまったことが示されています。細胞保持のこの問題に対処するための可能な見通しは、細胞または損傷部位に送達することができる治療薬のいずれかをカプセル化する生体適合性ヒドロゲル系を開発することです。このプロトコルにおける選択のヒドロゲルは、ポリ（エチレングリコール）は、細胞のカプセル化では、直前の使用広報によるメタクリレートでありますoceduresしかし、カプセル化することができる任意のヒドロゲルが8を使用してもよいです。直接損傷部位へのパッチの送達は、細胞が、下にある心筋に有益な因子を提供することができる時間の長さを増加させ、長期間にわたる細胞 - 組織接触を確実にします。 パッチアプローチのボトルネックは、心臓の表面にパッチを接着するのが困難です。多くのグループが、最も普及しているが、心臓の表面9,10に構造物を結びつけるための簡単な縫合糸である、種々の技術によってこれを克服しています。これは、構築物はより硬い材料で作られたケースの数は成功しますが、高い水濃度とパッチ構築物の繊細な性質のために、ヒドロゲル系にしようとしたときに失敗しました。これを克服するために、我々は、血栓形成の化学的性質を模倣するフィブリン糊外部接着剤系を利用しています。フィブリン糊は、INC、多数の医療手術で使用されています創傷シーラント11〜13として、製品の生体適合性を強調し、硬膜涙、気管支瘻、及び角膜移植をluding。さらに、フィブリンは、左心室破裂および冠動脈バイパス手術の外科的治療を含む心臓の目的の種々のために使用されている、しかし、心臓パッチの接着糊としてのその使用は、一般的に14〜17を使用していません。心臓の相互作用にパッチを確実にするために実行可能な接着システムを提供すること、外部心臓パッチの外側に直接配置することができる生体適合性の接着剤でトロンビンおよびフィブリノーゲン結果の単純な製剤。

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	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td>Harvard Model 687 Mouse Ventilator</td>
			<td>Harvard Apparatus</td>
			<td>55-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Inintech Biosciences LLC Dry Glass Bead Sterilizer</td>
			<td>Fisher Scientific</td>
			<td>NC9531961</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica MZ6 surgical microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cautery Kit</td>
			<td>Gemini</td>
			<td>GEM 5917</td>
			<td></td>
		</tr>
		<tr>
			<td>Delicate Forceps - 0.4mm Tips Angled</td>
			<td>Fine Science Tools</td>
			<td>11063-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Agricola Retractor - 3.5cm Spread</td>
			<td>Fine Science Tools</td>
			<td>17005-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring Scissors - 2.5mm Blades Straight</td>
			<td>Fine Science Tools</td>
			<td>15000-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Castroviejo Needle Holder - w/Lock Tungsten Carbide 14cm</td>
			<td>Fine Science Tools</td>
			<td>12565-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Scissors - Delicate Straight 10.5 cm</td>
			<td>Fine Science Tools</td>
			<td>14060-10</td>
			<td></td>
		</tr>
		<tr>
			<td>8-0 monofilament suture</td>
			<td>Ethicon</td>
			<td>8730P</td>
			<td></td>
		</tr>
		<tr>
			<td>6-0 Silk suture</td>
			<td>Ethicon</td>
			<td>639G</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>Sigma</td>
			<td>T7009</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen</td>
			<td>Sigma</td>
			<td>F3879</td>
			<td></td>
		</tr>
		<tr>
			<td>Vetbond Tissue Adhesive</td>
			<td>3M</td>
			<td>1469SB</td>
			<td></td>
		</tr>
	</tbody>
</table>

a hydrogel construct and fibrin-based glue approach to deliver therapeutics in a murine myocardial infarction model.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of treatments in vivo1. The traditional, established protocol has been further fine-tuned to minimize the damage to the animal. Notably, the pectoral muscle layers are teased away rather than simply cut, and the thoracotomy is approached intercostally as opposed to breaking the ribs in a sternotomy, preserving the integrity of the ribcage. With these changes, the overall stress on the animal is decreased. 
Stem cell therapies aimed to alleviate the damage caused by MIs have shown promise over the years for their pro-angiogenic and anti-apoptotic benefits. Current approaches of delivering cells to the heart surface typically involve the injection of the cells either near the damaged site, within a coronary artery, or into the peripheral blood stream2-4. While the cells have proven to home to the damaged myocardium, functionality is limited by their poor engraftment at the site of injury, resulting in diffusion into the blood stream5. This manuscript highlights a procedure that overcomes this obstacle with the use of a cell-encapsulated hydrogel patch. The patch is fabricated prior to the surgical procedure and is placed on the injured myocardium immediately following the occlusion of the left coronary artery. To adhere the patch in place, biocompatible external fibrin glue is placed directly on top of the patch, allowing for it to dry to both the patch and the heart surface. This approach provides a novel adhesion method for the application of a delicate cell-encapsulating therapeutic construct.

外科的処置の間に、左冠動脈の結紮は、閉塞した動脈の下流のブランチングマークすることによって同定することができます。結び目を作る前の試験のように、縫合糸は、それが適切な場所にあるかどうかを確認するために簡単に締め付けることができます。さらに、適切にビートに左心房の能力のほぼ瞬時減少動脈結果の閉塞ので、左心房は、システム内の血液の逆流に応じて拡大します?...

Watch this Scientific Journal Video about A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model. at JoVE.com

A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

ヒドロゲルは、構築し、マウス心筋梗塞モデルにおける治療薬を送達するためのフィブリンベースの接着剤のアプローチ。

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of ...

a-hydrogel-construct-fibrin-based-glue-approach-to-deliver

Research

JoVE Journal

Medicine

8.6K ビュー.  University of Illinois at Urbana-Champaign. This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

ビデオ: A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

Acute Myocardial Infarction in Rats

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evan's blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.

ラットにおける急性心筋梗塞

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

医学

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

心筋梗塞のマウスモデルにおける冠動脈結紮および心筋内注入

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ischemia and reperfusion. Immune response includes cytokine expression and release or secretion of endogenous ligands of innate immune receptors. Activation of innate immunity can potentially modulate infarct size. We have modified an existing murine closed-chest model using hanging weights which could be useful for studying myocardial pre- and postconditioning and the role of innate immunity in myocardial ischemia and reperfusion. This model allows animals to recover from surgical trauma before onset of myocardial ischemia.
Volatile anesthetics have been intensely studied and their preconditioning effect for the ischemic heart is well known. However, this protective effect precludes its use in open chest models of coronary artery ligation. Thus, another advantage could be the use of the well controllable volatile anesthetics for instrumentation in a chronic closed-chest model, since their preconditioning effect lasts up to 72 hours. Chronic heart diseases with intermittent ischemia and multiple hit models are other possible applications of this model.
For the chronic closed-chest model, intubated and ventilated mice undergo a lateral blunt thoracotomy via the 4th intercostal space. Following identification of the left anterior descending a ligature is passed underneath the vessel and both suture ends are threaded through an occluder. Then, both suture ends are passed through the chest wall, knotted to form a loop and left in the subcutaneous tissue. After chest closure and recovery for 5 days, mice are anesthetized again, chest skin is reopened and hanging weights are hooked up to the loop under ECG control.
At the end of the ischemia/reperfusion protocol, hearts can be stained with TTC for infarct size assessment or undergo perfusion fixation to allow morphometric studies in addition to histology and immunohistochemistry.

Surgical trauma induces an inflammatory response. Cytokines and endogenous ligands are known to modulate myocardial infarct size following ischemia and reperfusion. We present a modified closed-chest model of murine ischemia and reperfusion using hanging weights to minimize effects of thoracotomy.

心筋虚血再灌流障害のマウスクローズド胸モデル

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

豚における心筋梗塞および機能的結果の評価

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are therefore required to confine cardiac damage, promote survival and reduce the disease burden of heart failure. Large animal experiments are an essential part in the translational process from experimental to clinical therapies. To optimize clinical translation, robust and representative outcome measures are mandatory. The present manuscript aims to address this need by describing the assessment of three clinically relevant outcome modalities in a pig acute myocardial infarction (AMI) model: infarct size in relation to area at risk (IS/AAR) staining, 3-dimensional transesophageal echocardiography (TEE) and admittance-based pressure-volume (PV) loops. Infarct size is the main determinant driving the transition from AMI to heart failure and can be quantified by IS/AAR staining. Echocardiography is a reliable and robust tool in the assessment of global and regional cardiac function in clinical cardiology. Here, a method for three-dimensional transesophageal echocardiography (3D-TEE) in pigs is provided. Extensive insight into cardiac performance can be obtained by admittance-based pressure-volume (PV) loops, including intrinsic parameters of myocardial function that are pre- and afterload independent. Combined with a clinically feasible experimental study protocol, these outcome measures provide researchers with essential information to determine whether novel therapeutic strategies could yield promising targets for future testing in clinical studies.

Reliable and accurate outcome assessment is the key for translation of preclinical therapies into clinical treatment. The current paper describes how to assess three clinically relevant primary outcome parameters of cardiac performance and damage in a pig acute myocardial infarction model.

急性心筋梗塞の豚モデルにおける主要アウトカム評価

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

新生児マウスの心筋梗塞、心臓再生のモデル

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

Murine Left Anterior Descending (LAD) Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction

Ischemic heart disease (IHD), or acute coronary syndrome (ACS), is one of the leading causes of death in the United States. IHD is characterized by reduced blood supply to the heart, resulting in the loss of oxygen to and the ensuing necrosis of the heart muscle. The MI model has gained popularity for its use as a short-term ischemia-reperfusion model and a long-term permanent ligation model. Below, we describe a reliable method for the permanent ligation of the LAD. With mouse genetic engineering technology becoming more advanced, and with an increasing availability of quality murine surgical instruments, the mouse has become a popular model for MI surgeries. Our surgical model incorporates the use of an easily reversible anesthetic for the rapid recovery of the mouse; a minimally invasive endotracheal intubation without involving a tracheotomy; and a thoracentesis through the original thoracotomy site without creating an additional incision in the chest, as is done in some other methods, to effectively remove excess blood and air from the chest cavity. This method is comparatively less invasive than other methods, which dramatically reduces surgical and post-surgical complications and mortality and improves reproducibility.

Murine Left Anterior Descending LAD Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction

We provide a reliable method for left anterior descending artery (LAD) ligation in a mouse model. This method is comparatively less invasive than other methods, involving endotracheal intubation, a left-sided thoracotomy approach, and thoracentesis. This method can be used as a model for both acute and chronic myocardial infarction (MI).

マウス左前方降順（LAD）冠動脈結紮：心筋梗塞のための改善と簡略化されたモデル

Ischemic heart disease (IHD), or acute coronary syndrome (ACS), is one of the leading causes of death in the United States. IHD is characterized by ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

左前皮下冠動脈の永久的なライゲーションを用いたマウス心筋梗塞モデル

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

マウスの心筋梗塞を研究する凍結傷害モデル

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...

Establishing a Swine Model of Post-myocardial Infarction Heart Failure for Stem Cell Treatment

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major causes of mortality and morbidity around the world. Cell-based therapies for cardiac repair and improvement of left ventricular function after MI have attracted considerable attention. Accordingly, the safety and efficacy of these cell transplantations should be tested in a preclinical large animal model of HF prior to clinical use. Pigs are widely used for cardiovascular disease research due to their similarity to humans in terms of heart size and coronary anatomy. Therefore, we sought to present an effective protocol for the establishment of a porcine chronic HF model using closed-chest coronary balloon occlusion of the left circumflex artery (LCX), followed by rapid ventricular pacing induced with pacemaker implantation. Eight weeks later, the stem cells were administered by intramyocardial injection in the peri-infarct area. Then the infarct size, cell survival, and left ventricular function (including echocardiography, hemodynamic parameters, and electrophysiology) were evaluated. This study helps establish a stable preclinical large animal HF model for stem cell treatment.

We sought to establish a swine model of heart failure induced by left circumflex artery blockage and rapid pacing to test the effect and safety of intramyocardial administration of stem cells for cell-based therapies.

幹細胞治療のための心筋梗塞後心不全の豚モデルの確立

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major ...

Methods Article

ヒドロゲルは、構築し、マウス心筋梗塞モデルにおける治療薬を送達するためのフィブリンベースの接着剤のアプローチ。